1. Field of the Invention
This invention is directed to preparation and expression of synthetic genes encoding polypeptides containing protective epitopes of botulinum neurotoxin (BoNT). The invention is also directed to methods of vaccination against botulism using the expressed peptides.
2. Related Art
The sporulating, obligate anaerobic, gram-positive bacillus Clostridium produces eight forms of antigenically distinct exotoxins. Tetanus neurotoxin (TeNT) is produced by Clostridium tetani while Clostridium botulinum produces seven different neurotoxins which are differentiated serologically by specific neutralization. The botulinum neurotoxins (BoNT) have been designated as serotypes A, B, C1, D, E, P, and G. Botulinum neurotoxins (BoNT) are the most toxic substances known and are the causative agents of the disease botulism. BoNT exert their action by inhibiting the release of the neurotransmitter acetylcholine at the neuromuscular junction (Habermann, E., et al., (1986), “Clostridial Neurotoxins: Handling and Action at the Cellular and Molecular Level,” Cur. Top. Microbiol. Immunol., 129:93–179; Schiavo, G., et al., (1992a), “Tetanus and Botulinum-B Neurotoxins Block Neurotransmitter Release by Proteolytic Cleavage of Synaptobrevin,” Nature, 359:832–835; Simpson, L. L., (1986), “Molecular Pharmacology of Botulinum Toxin and Tetanus Toxin,” Annu. Rev. Pharmacol. Toxicol., 26:427–453) which leads to a state of flaccid paralysis. Indeed, only a few molecules of toxin can abolish the action of a nerve cell. Polyclonal antibodies derived for a specific neurotoxin can neutralize the toxic effects of that toxin but will not cross-neutralize another toxin serotype. Thus, to protect against all seven toxins, one needs seven vaccines.
Botulinum neurotoxins are translated as a single 150 kDa polypeptide chain and then posttranslationally nicked, forming a dichain consisting of a 100 kDa heavy chain and a 50 kDa light chain which remain linked by a disulfide bond (DasGupta, B. R., et al., (1972), “A Common Subunit Structure in Clostridium botulinum Type A, B, and E Toxins,” Biophys. Res. Commun., 48:108–112; DasGupta, B. R., (1989), “The Structure of Botulinum Neurotoxins,” Botulinum Neurotoxin and Tetanus Toxin, (Simpson, L. L., Ed.), pp. 53–67, Academic Press, New York). Most of the clostridial strains contain specific endogenous proteases which activate the toxins at a protease-sensitive loop located approximately one third of the way into the molecule from the amino-terminal end. Upon reduction and fractionation (electrophoretically or chromatographically), the two chains can be separated; one chain has a Mr of ˜100 kDa and is referred to as the heavy chain while the other has a Mr ˜50 kDa and is termed the light chain.
The mechanism of nerve intoxication is accomplished through the interplay of three key events, each of which is performed by a separate portion of the neurotoxin protein. First, the carboxy half of the heavy chain (fragment C or HC is required for receptor specific binding to cholinergic nerve cells (Black, J. D., et al., (1986), “Interaction of 125I-botulinum Neurotoxins with Nerve Terminals. I. Ultrastructural Autoradiographic Localization and Quantitation of Distinct Membrane Acceptors for Types A and B on Motor Nerves,” J. Cell Biol., 103:521–534; Nishiki, T.-I., et al., (1994), “Identification of Protein Receptor for Clostridium botulinum Type B Neurotoxin in Rat Brain Synaptosomes,” J. Biol. Chem., 269:10498–10503; Shone, C. C., et al., (1985), “Inactivation of Clostridium botulinum Type A Neurotoxin by Trypsin and Purification of Two Tryptic Fragments. Proteolytic Action Near the COOH-terminus of the Heavy Subunit Destroys Toxin-Binding Activity, Eur. J. Biochem., 151:75–82). There is evidence suggesting that polysialogangliosides (van Heyningen, W. E., (1968), “Tetanus,” Sci. Am., 218:69–77) could act as receptors for the toxins but the data supporting a specific receptor remains equivocal (Middlebrook, J. L., (1989), “Cell Surface Receptors for Protein Toxins,” Botulinum Neurotoxins and Tetanus Toxin, (Simpson, L. L., Ed.) pp. 95–119, Academic Press, New York). After binding, the toxin is internalized into an endosome through receptor-mediated endocyctosis (Shone, C. C., et al., (1987), “A 50-kDa Fragment from the NH2-tenninus of the Heavy Subunit of Clostridium botulinum Type A Neurotoxin Forms Channels in Lipid Vesicles, Euro. J. Biochem., 167:175–180). The amino terminal half of the heavy chain is believed to participate in the translocation mechanism of the light chain across the endosomal membrane (Simpson, 1986; Poulain, B., et al., (1991), “Heterologous Combinations of Heavy and Light Chains from Botulinum Neurotoxin A and Tetanus Toxin Inhibit Neurotransmitter Release in Aplysia,” J. Biol. Chem., 266:9580–9585; Montal, M. S., et al., (1992), “Identification of an Ion Channel-Forming Motif in the Primary Structure of Tetanus and Botulinum Neurotoxins,” FEBS, 313:12–18). The low pH environment of the endosome may trigger a conformational change in the translocation domain, thus forming a channel for the light chain The final event of intoxication involves enzymatic activity of the light chain, a zinc-dependent endoprotease (Schiavo, 1992a; Schiavo, G., et al., (1992b), “Tetanus Toxin is a Zinc Protein and its Inhibition of Neurotransmitter Release and Protease Activity Depend on Zinc,” EMBO J., 11:3577–3583), on key synaptic vesicle proteins (Schiavo, 1992a; Oguma, K., et al., (1995), “Structure and Function of Clostridium botulinum Toxins,” Microbiol. Immunol., 39:161–168; Schiavo, G., et al., (1993), “Identification of the Nerve Terminal Targets of Botulinum Neurotoxin Serotypes A, D, and E,” J. Biol. Chem., 268:23784–23787; Shone, C. C., et al., (1993), “Proteolytic Cleavage of Synthetic Fragments of Vesicle-Associated Membrane Protein, Isoform-2 by Botulinum Type B Neurotoxin,” Eur. J. Biochem., 217:965–971) necessary for neurotransmitter release. The light chains of BoNT serotypes A, C1, and E cleave SNAP-25 (synaptosomal-associated protein of M25,000), serotypes B, D, F, and G cleave VAMP/synaptobrevin (synaptic vesicle-associated membrane protein); and serotype C1 cleaves syntaxin. Inactivation of SNAP-25, VAMP, or syntaxin by BoNT leads to an inability of the nerve cells to release acetylcholine resulting in neuromuscular paralysis and possible death, if the condition remains untreated.
Human botulism poisoning is generally caused by type A, B, E or rarely, by type F toxin. Type A and B are highly poisonous proteins which resist digestion by the enzymes of the gastrointestinal tract. Foodborne botulism poisoning is caused by the toxins present in contaminated food, but wound and infant botulism are caused by in vivo growth in closed wounds and the gastrointestinal tract respectively. The toxins primarily act by inhibiting the neurotransmitter acetylcholine at the neuromuscular junction, causing paralysis. Another means for botulism poisoning to occur is the deliberate introduction of the toxin(s) into the environment as might occur in biological warfare. When the cause of botulism is produced by toxin rather than by in vivo infection the onset of neurologic symptoms is usually abrupt and occurs within 18 to 36 hours after ingestion. The most common immediate cause of death in respiratory failure due to diaphragmatic paralysis. Home canned foods are the most common sources of toxins. The most frequently implicated toxin is toxin A, which is responsible for more than 50% of morbidity resulting from botulinum toxin.
Because even small amounts of botulinal toxin can cause serious illness, persons such as laboratory workers who are exposed to toxin must learn to handle all samples that may contain toxin with extreme care. It is also suggested that such workers be protected from illness by vaccination against the toxins. Furthermore, persons exposed to conditions in which botulism toxins might be in the environment which might be inhaled or ingested, such as military personnel, need to be protected from the toxin.
Agents that abolish the action of BoNT have been investigated since the 1940s. Early work at Fort Detrick in the 1940s lead to the development of a toxoid vaccine to protect against serotypes A, B, C1, D, and E toxins. The toxoid vaccine was manufactured by growing five Clostridium botulinum strains, extracting and precipitating the toxin from the growth media after cell lysis. Formalin was added to the crude preparation to inactivate the neurotoxin. Residual formalin was left in the vaccine product to ensure the toxin remains non-toxic. The product was adsorbed to aluminum hydroxide and blended. Currently, a pentavalent toxoid vaccine against serotypes A through E (Anderson, J. H., et al., (1981), “Clinical Evaluation of Botulinum Toxoids,” Biomedical Aspects of Botulism, (Lewis, G. E., Ed.), pp. 233–246, Academic Press, New York; Ellis, R. J., (1982), “Immunobiologic Agents and Drugs Available from the Centers for Disease Control. Descriptions, Recommendations, Adverse Reactions and Scrologic Response,” 3rd ed., Centers for Disease Control. Atlanta, Ga.; Fiock, M. A., et al., (1963), “Studies of Immunities to Toxins of Clostridium Botulinum. IX. Immunologic Response of Man to Purified Pentavalent ABCDE Botulinum Toxoid,” J. Immunol., 90:697–702; Siegel, L. S., (1988), “Human Immune Response to Botulinum Pentavalent (ABCDE) Toxoid Determined by a Neutralization Test and by an Enzyme-Linked Immunosorbent Assay,” J. Clin. Microbiol., 26:2351–2356), available under Investigational New Drug (IND) status, is used to immunize specific populations of at-risk individuals, i.e., scientists and health care providers who handle BoNT and our armed forces who may be subjected to weaponized forms of the toxin Though serotypes A, B, and E are most associated with botulism outbreaks in humans, type F has also been diagnosed (Midura, T. F., et al., (1972), “Clostridium botulinum Type F: Isolation from Venison Jerky,” Appl. Microbiol., 24:165–167; Green, J., et al., (1983), “Human Botulism (Type F)—A Rare Type,” Am. J. Med., 75:893–895; Sonnabend, W. F., et al., (1987), “Intestinal Toxicoinfection by Clostridium botulinum Type F in an Adult. Case Associated with Guillian-Barre Syndrome,” Lancet, 1:357–361; Hatheway, C. L., (1976), “Toxoid of Clostridium botulinum Type F: Purification and Immunogenicity Studies,” Appl. Environ. Microbiol., 31:234–242). A separate monovalent toxoid vaccine against BoNTF is available under IND status Hatheway demonstrated that the BoNTF toxoid could protect guinea pigs against a homologous challenge (Wadsworth, J. D. F., et al., (1990), “Botulinum Type F Neurotoxin,” Biochem. J., 268:123–128).
Even though toxoid vaccines are available, there are numerous shortcomings with their current use and ease of production. First, because C. botulinum is a spore-former, a dedicated facility is required to manufacture a toxin-based product. The requirement for a dedicated manufacturing facility is not trivial. It is extremely costly to renovate and upgrade an existing facility or to build a new one and then to maintain the facility in accordance with current Good Manufacturing Practices (cGMP) to manufacture one vaccine. Second, the yields of toxin production from C. botulinum are relatively low. Third, the toxoiding process involves handling large quantities of toxin and thus is dangerous, and the added safety precautions increase the cost of manufacturing. Fourth, the toxoid product for types A–E consists of a crude extract of clostridial proteins that may influence immunogenicity or reactivity of the vaccine, and the type F toxoid is only partially purified (IND 5077). Fifth, because the toxoiding process involves the use of formaldehyde, which inactivates the toxin, and residual levels of formaldehyde (not to exceed 0.02%) are part of the product formulation to prevent reactivation of the toxin, the vaccine is reactogenic. An additional component of the toxoid vaccines is the preservative thimerosal (0.01%), which also increases the reactogenicity of the product.
The development of a new-generation, recombinant vaccine could alleviate many of the problems associated with the toxoid. A recombinant vaccine would eliminate the need for a dedicated manufacturing facility. Presently, many cgMP facilities are in existence and available that could manufacture a recombinant product. There would be no need to culture large quantities of a hazardous toxin-producing bacterium. Production yields from a genetically engineered product is expected to be high. There would be no need to treat the vaccine with formalin because the product would be non-toxic from the outset. Recombinant products would be purer, less reactogenic, and more fully characterized. Thus, the cost of a recombinant product would be expected to be much lower than a toxoid because there would be no expenditures required to support a dedicated facility, and the higher production yields would reduce the cost of the vaccine product.